Enterococcus Faecalis LTA Induces TNF-α through NF-κB and p38 Pathways

Objectives: Enterococcus faecalis, a Gram-positive bacterium, is considered a major pathogen related to refractory root canal infection and endodontic failure. D-alanyl lipoteichoic acid (LTA) is a principal component of bacterial membrane and a major virulence factor of E. faecalis. This purposes of the present sstudy were to investigate the immune-inflammatory response and signaling mechanism of human monocytes to purified E. faecalisLTA. Methods: Highly purified intact LTA was obtained from a highly virulent E. faecalis clinical strain P25RC by n-butanol extraction and hydrophobia interaction chromatography (HIC) purification. The fractions containing LTA were determined by phosphate detection and lyophilized. Contaminations with LPS and proteins were excluded by LAL assay and SDS-PAGE electrophoresis, respectively. Structurally intact LTA was analyzed by nuclear magnetic resonance (NMR). Before LTA stimulation assays, THP-1 monocytes were pretreated with phorbol 12-myristate 13-acetate (PMA) to differentiate into macrophages. Cells were treated with LTA in concentration gradients. The transcript expression of TLR2, CD14, MyD88, TIRAP and IRAK1 was evaluated by qPCR. The cytokines TNF-α and IL-10 were quantified using ELISA. The activated and total NF-κB p65 and three MAP kinases (p38, ERK1/2 and JNK) were assessed using western blot. Results: D-alanyl LTA induced the gene expression of TLR2 and MyD88 whilst down-regulated CD14 suggesting a TLR2-dependent and CD14-independent immune-inflammatory activity.  LTA stimulated the expression of pro-inflammatory cytokine TNF-α, but not the anti-inflammatory cytokine IL-10, possibly through NF-κB and p38 pathways as shown by the activated the phosphorylation in western blot analysis. Conclusion: We for the first time have shown that E. faecalis LTA induce the immune-inflammatory response of human monocytes via TLR-2 dependent NF-κB and p38 signal transduction. These data provide a new insight on endodontic infection for targeting E. faecalis LTA as a therapeutic approach (Supported by RFCID/HHSRF Grant No.12110772).